CN102186457B - Skin-whitening composition comprising an extract, fraction or compound derived from lindera erythrocarpa - Google Patents

Skin-whitening composition comprising an extract, fraction or compound derived from lindera erythrocarpa Download PDF

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CN102186457B
CN102186457B CN200980141207.8A CN200980141207A CN102186457B CN 102186457 B CN102186457 B CN 102186457B CN 200980141207 A CN200980141207 A CN 200980141207A CN 102186457 B CN102186457 B CN 102186457B
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fructus
compound
linderae glaucae
extract
chemical formula
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CN102186457A (en
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金基玉
高丽暻
晋荣晙
康瑉哲
韩宗宪
崔昊旻
高光晓
金俸奭
晋浩景
秦炅希
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JEJU HI TECH INDUSTRY DEV I
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat

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Abstract

The present invention relates to a skin-whitening composition comprising an extract, a fraction or a compound derived from Lindera erythrocarpa. More specifically, the present invention relates to a skin-whitening composition comprising having an active ingredient consisting of an ethanol extract obtained by using ethanol to extract Lindera erythrocarpa, a solvent fraction obtained therefrom, or a compound isolated and purified therefrom.

Description

The skin whitening composition that comprises the extract, part or the compound that are derived from Fructus Pyracanthae Fructus Linderae Glaucae
Technical field
The present invention relates to comprise the skin whitening composition of the extract, part or the compound that are derived from Fructus Pyracanthae Fructus Linderae Glaucae (Lindera erythrocarpa).More specifically, the present invention relates to comprise the ethanol extraction from Fructus Pyracanthae Fructus Linderae Glaucae as active component, from the skin whitening composition of the solvent part of described ethanol extraction or the compound of and purification separated from described ethanol extraction.
Background technology
The physiologically active compound from natural product is paid close attention in a large amount of research.Plant resources is widely used in treatment and the prevention of disease or assists as healthy.For example, alpha-tocopherol, vitamin C, carotenoid and flavonoid are as natural antioxidant.In the animal and plant of wide region, found to have the compound of antioxidant activity, particularly a large amount of research is from plant.In body, experiment shows to suppress or remove from the secondary metabolite of plant the generation of free radical and active oxygen, thus the cell injury of anti-oxidation induction.
The naturally occurring antioxidant of major part of reporting is up to now from plant and be mainly polyphenolic substance.Particularly, flavonoid has the function that stops lipid oxidation and oxidative stress and Scavenger of ROS, thus prevention or slow down aging, blocking-up cancer and cardiopathic generation or progress.Thus, flavonoid has been used to various fields, comprises food industry, pharmaceuticals industry and cosmetics industry.In addition, melanin is at the ubiquitous polymerization phenols of nature pigment, and it sees and comprises animal, plant and most of biologies of microorganism even, and as the important thing against sunshine (photoprotectant) of protection body antagonism UV radiation.
Melanin strengthens the survival ability of antagonism UV radiation, dry and extreme temperature, and has strengthened the quality of coffee, tea and cigar.On the other hand, the excessive melanic synthetic known generation that can cause generation, the promotion skin aging of body freckle and speckle and participate in skin carcinoma.The plurality of enzymes that comprises tryrosinase regulates melanic biosynthesis.Among these enzymes, tryrosinase is responsible for the early stage oxidation of dihydroxy indole afterwards of biosynthesis, and wherein tyrosine is converted into L-3,4 dihydroxyphenylalanine quinine.In this, the research of searching tyrosinase inhibitor occupies part and parcel in the exploitation of skin whitener.The tyrosinase inhibitor of exploitation comprises hydroquinone, 4-hydroxyanisol, ascorbic acid derivates, kojic acid, Azelaic Acid, corticosteroid, biostearin, arbutin, catechin and 3,4,5-trimethoxy cinnamic acid Herba thymi vulgaris phenolic ester at present.Yet, because the problem of their safety and economic benefit is difficult to use them.
Fructus Pyracanthae Fructus Linderae Glaucae is the plant that belongs to Lauraceae (Lauraceae family).Known 45 genus and 1500 kinds that have Lignum cinnamomi camphorae in the whole world, and there are 6 genus and 12 kinds in Korea S.In Korea S, no matter be male or female Fructus Pyracanthae Fructus Linderae Glaucae, great majority were outputed lurid flower and are born in JIUYUE the erythrocarpus that size is about 8mm in April to May.Fructus Pyracanthae Fructus Linderae Glaucae is the deciduous tree that about 5m is high and originates in Korea S south, Japan and Chinese warm area.The dry fruit of tree has distinctive fragrance and bitter in the mouth, in Japan, they is used as to stomach invigorating medicine and neuralgic analgesic.It is reported, the leaf of Fructus Pyracanthae Fructus Linderae Glaucae and the ethanol extraction of fruit and lucidone (lucidone) (a kind of cyclopentanedione) have potential anti-inflammatory activity (Wang etal., 2008).The known cyclopentanedione compound from the separation of Fructus Pyracanthae Fructus Linderae Glaucae has active anticancer (Ohet al., 2005; 10-0658519 Korean Patent).
Considering after background technology comprehensively, the inventor has obtained the ethanol extraction of the Fructus Pyracanthae Fructus Linderae Glaucae that originates in Korea S's Jizhou Island and follow-up solvent part thereof and from cyclopentanedione compound and the cyclopentanedione derivant of described solvent part, in B16F10 mouse melanin tumor cell and RAW264.7 cell, detect antioxidant activity and the inhibition activity to melanin biosynthesis of above material, and finally find that above material can be used as the functional activity material of skin whitening cosmetics, food and pharmaceutical composition, thereby complete the present invention.
Disclosure
Technical problem
Therefore, the object of this invention is to provide and comprise as the extract of Fructus Pyracanthae Fructus Linderae Glaucae of active component, the solvent of described extract part or from the separated cyclopentanedione compound of described solvent part or the skin whitening composition of derivant.
Technical scheme
According to an aspect of the present invention, the invention provides and comprise as the extract of Fructus Pyracanthae Fructus Linderae Glaucae of active component or the skin whitening composition of the solvent of described extract part.
In the present invention, for obtain the solvent of extract from Fructus Pyracanthae Fructus Linderae Glaucae, be selected from water, such as the lower alcohol of methanol or ethanol and above combination, and preferred alcohol.According to the present invention, from extracting the diluent of the extract solution obtain, extract solution or concentrated solution, the residue obtaining by dry extract solution, all falling in the scope of extract from the material of the thick purification of extract solution with from the material of the purification of extract solution.
In embodiments of the invention, can be prepared as follows the extract from Fructus Pyracanthae Fructus Linderae Glaucae: with hot blast, Fructus Pyracanthae Fructus Linderae Glaucae is dry, dry Fructus Pyracanthae Fructus Linderae Glaucae pulverized, under room temperature, the Fructus Pyracanthae Fructus Linderae Glaucae of pulverizing immersed in 70% ethanol three days and follow and stir exudate to be provided, exudate is filtered and concentrating under reduced pressure filter liquor.
In embodiments of the invention, can be prepared as follows the solvent part from the extract of Fructus Pyracanthae Fructus Linderae Glaucae: the extract from Fructus Pyracanthae Fructus Linderae Glaucae is suspended in distilled water, and with the normal hexane (n-Hex), the dichloromethane (CH that come from non-polar solven 2cl 2), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) be suspension part.
In one embodiment of the invention, be prepared as follows each the solvent part from the extract of Fructus Pyracanthae Fructus Linderae Glaucae: the ethanol extraction of Fructus Pyracanthae Fructus Linderae Glaucae is suspended in distilled water, uses separatory funnel, utilize normal hexane (n-Hex), dichloromethane (CH from non-polar solven 2cl 2), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) be extract part, filter and by gained part concentrating under reduced pressure.
According to a further aspect in the invention, the invention provides at least one that comprise as active component and be selected from the skin whitening composition of the compound in the represented compound of following Chemical formula 1 to 10:
Chemical formula 1
Chemical formula 2
Chemical formula 3
Chemical formula 4
Chemical formula 5
Chemical formula 6
Chemical formula 7
Chemical formula 8
Chemical formula 9
Chemical formula 10
In order to use in the present invention, can be from Fructus Pyracanthae Fructus Linderae Glaucae the separated and above compound of purification.Or, can obtain all above-claimed cpds except compound 10 by commercially available prod, or can synthesize them by known synthetic method.
According to the preferred embodiment of the invention, compositions of the present invention is cosmetics preparation.
Except the extract from Fructus Pyracanthae Fructus Linderae Glaucae, the solvent of extract partly or from the compound of extract separation is also used as active component, cosmetics compositions of the present invention can comprise conventional cosmetics composition, for example conventional adminicle and carrier, for example antioxidant, stabilizing agent, solubilizing agent, vitamin, coloring agent and aromatic.Above cosmetics compositions can also comprise the skin absorption promoter for the cosmetic result of enhanced activity composition.
Can be by the conventional any preparation in cosmetics compositions preparation cost of the present invention field.For example, can be mixed with solution, suspension, emulsion, paste, gel, emulsifiable paste, lotion, powder, soap, be contained cleaning agent, oil, foundation cream, foundation emulsion, foundation cream wax and the spray of surfactant, but be not limited to this.The instantiation of preparation comprises astrigent lotion, nutrition washing liquid, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleaning foam, clean water, facial film, spray or powder.
When this compositions is formulated into paste, emulsifiable paste or gel, can be by animal oil, vegetable oil, wax, paraffin, starch, Tragacanth, cellulose derivative, Polyethylene Glycol, silicon, bentonite, silicon dioxide, Talcum or zinc oxide as carrier.
When preparation is the form of powder or spray, can be by lactose, Talcum, silicon dioxide, aluminium hydroxide, calcium silicates or polyamide powder as carrier.Especially, the in the situation that of spray, can also comprise the propellant such as Chlorofluorocarbons (CFCs), propane/butane or dimethyl ether.
For the preparation of solution or emulsion form, can be by solvent, solubilizing agent or emulsifying agent as carrier.The example of carrier comprises: water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1, the fatty acid ester of 3-butyl glycol oil, glycerin fatty ester, Polyethylene Glycol or sorbitan.
Preparation for form of suspension, carrier can comprise the liquid diluent such as water, ethanol or propylene glycol, such as the suspending agent of ethoxylation isooctadecanol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan esters, microcrystalline Cellulose, aluminium hydroxide oxide (aluminummethahydroxide), bentonite, agar or Tragacanth etc.
For the preparation of the cleaning agent form containing surfactant, carrier can comprise fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoesters, isethionate, imdazole derivatives, methyl tauride, sarcosinate, fatty acyl amidogen ether sulfuric ester, alkyl amido betaine, fatty alcohol, fatty glyceride, fatty diglycollic amide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester etc.
According to the preferred embodiments of the invention, compositions of the present invention is pharmaceutical preparation.
When being formulated as pharmaceutical preparation, compositions of the present invention can comprise pharmaceutically acceptable carrier.The normally used pharmaceutically acceptable carrier in this area can be included in pharmaceutical composition.The example of the pharmaceutically acceptable carrier using in compositions comprises lactose, glucose, sucrose, Sorbitol, mannitol, starch, arabic gum, calcium phosphate, alginate, gelatin, calcium silicates, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, Talcum, magnesium stearate and mineral oil, but is not limited to this.In addition to these, pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweeting agent, flavoring agent, emulsifying agent, suspending agent and/or antiseptic.
Can give pharmaceutical composition of the present invention by various oral or parenteral route, for example, oral administration or percutaneous dosing.Yet, preferably by giving pharmaceutical composition by parenteral route, percutaneous dosing particularly, and more preferably by local application.
According to comprising dosage form, administering mode, age, body weight, sex, the patient's that treats disease condition and the many factors of diet, administration time, route of administration, excretion rate and reaction sensibility, when pharmaceutical composition of the present invention is used, when spraying or administration, its suitable dosage can be different.When making oral formulations, can with the every daily dose of adult of 5mg/kg to 30mg/kg and the every daily dose of adult that is preferably 10mg/kg, carry out single dose administration every day or be divided into a plurality of dosage carrying out administration by the solvent part of the extract of Fructus Pyracanthae Fructus Linderae Glaucae, this extract or from the separated compound of this solvent part.For adult's local application, can carry out with every daily dose of 1.0ml to 3.0ml single dose and use and maybe can be divided into five dosage of as many as and use, continue to use at least one month.
According to well known to a person skilled in the art method, pharmaceutical composition of the present invention can be formulated as unit dosage forms and maybe can be included in multiple-unit container together with pharmaceutically acceptable carrier and/or excipient.Under this background, pharmaceutical composition can be formulated as the peroral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol etc., such as topical formulations or the applicable form of any pharmacy of ointment, emulsifiable paste etc., and can comprise dispersant or stabilizing agent.
In the preferred embodiment of the invention, compositions of the present invention is food formulation.
When food prepared therefrom preparation, except as active component from the extract of Fructus Pyracanthae Fructus Linderae Glaucae, the part of this extract or from the compound of this extract separation, compositions of the present invention comprises prepares the composition that food adds conventionally, for example protein, carbohydrate, lipid, nutrient, flavoring agent and flavorant.The example of carbohydrate comprises the monosaccharide such as glucose and fructose, such as the disaccharide of maltose, sucrose, and oligosaccharide, such as the polysaccharide of dextrin, cyclodextrin etc., and such as the sugar alcohol of xylitol, Sorbitol, erythritol etc.Flavorant can be natural [thaumatin (thaurmatin), Stevia rebaudiana (Bertoni) Hemsl extract (for example, RA, glycyrrhizin etc.)] and synthetic (glucide, aspartame etc.).
According to other aspects of the invention, the invention provides the represented compound of following Chemical formula 10:
[Chemical formula 1 0]
In the present invention, can be from Fructus Pyracanthae Fructus Linderae Glaucae and the preferred compound of separation chemistry formula 10 from the bark of Fructus Pyracanthae Fructus Linderae Glaucae.
According to other aspects of the invention, the invention provides the method for the compound of separation chemistry formula 10 from Fructus Pyracanthae Fructus Linderae Glaucae.
[Chemical formula 1 0]
In preferred embodiments, the method for the compound of separation chemistry formula 10 can comprise the following steps:
Fructus Pyracanthae Fructus Linderae Glaucae is immersed in solvent so that the extract from Fructus Pyracanthae Fructus Linderae Glaucae to be provided, and described solvent is selected from water, C 1to C 4lower alcohol or its combination;
With hexane, dichloromethane, ethyl acetate and butanols by described Fructus Pyracanthae Fructus Linderae Glaucae extract part, so that minute other solvent part to be provided;
Use hexane and ethyl acetate gradient as eluent, described Fructus Pyracanthae Fructus Linderae Glaucae solvent is partly carried out to elementary silica gel column chromatography, so that chromatography part to be provided; And
Use the mixture of hexane and ethyl acetate as eluent, the part of elementary silica gel column chromatography is carried out to purification by secondary silica gel column chromatography, so that the polymethoxy phenolic compounds of new Chemical formula 10 to be provided.
In the method for the invention, " Fructus Pyracanthae Fructus Linderae Glaucae " refers to the bark of Fructus Pyracanthae Fructus Linderae Glaucae.
In the present invention, can use and be selected from following solvent and obtain Fructus Pyracanthae Fructus Linderae Glaucae extract: water, such as the C of methanol or ethanol 1to C 4lower alcohol or above combination, or preferably use ethanol.In the present invention, extract comprise be selected from following any: the extract solution obtaining by extraction, the diluent of extract solution or concentrated solution, the residue obtaining by dry extract solution, from the material of the thick purification of extract solution or from the material of the purification of extract solution.
According to embodiment of the present invention, can obtain as follows Fructus Pyracanthae Fructus Linderae Glaucae extract: with hot blast, Fructus Pyracanthae Fructus Linderae Glaucae is dry, dry Fructus Pyracanthae Fructus Linderae Glaucae pulverized, under room temperature, the Fructus Pyracanthae Fructus Linderae Glaucae of pulverizing immersed in 70% ethanol three days and follow and stir exudate to be provided, exudate is filtered and concentrating under reduced pressure filter liquor.
In embodiments of the invention, can be prepared as follows the solvent part of Fructus Pyracanthae Fructus Linderae Glaucae extract: Fructus Pyracanthae Fructus Linderae Glaucae extract is suspended in distilled water, uses normal hexane (n-Hex), dichloromethane (CH from non-polar solven 2cl 2), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) be suspension part.
In embodiments of the invention, can be prepared as follows each solvent part of Fructus Pyracanthae Fructus Linderae Glaucae extract: the ethanol extraction of Fructus Pyracanthae Fructus Linderae Glaucae is suspended in distilled water, uses separatory funnel, utilize normal hexane (n-Hex), dichloromethane (CH from non-polar solven 2cl 2), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) be extract part, filter and concentrating under reduced pressure so that minute other solvent part to be provided.
Useful effect
The skin whitening composition of the extract based on from Fructus Pyracanthae Fructus Linderae Glaucae, part or compound comprises the cyclopentanedione or derivatives thereof as active component, its show to the synthetic inhibition activity of melanin higher than conventional skin whitener arbutin.
Accompanying drawing explanation
Fig. 1 schematically illustrates the flow chart that obtains the process of ethanol extraction and solvent part thereof from Fructus Pyracanthae Fructus Linderae Glaucae.
Fig. 2 shows the HPLC analytical data of Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction of the present invention.
Fig. 3 shows the HPLC analytical data of the normal hexane part that is derived from Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction.
Fig. 4 shows the HPLC analytical data of the dichloromethane part that is derived from Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction.
Fig. 5 shows the HPLC analytical data of the ethyl acetate part that is derived from Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction.
Fig. 6 shows the HPLC analytical data of the n-butyl alcohol part that is derived from Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction.
Fig. 7 shows the HPLC analytical data of the water section that is derived from Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction.
Fig. 8 shows the HPLC data for compound 1 quantitative analysis.
Fig. 9 shows compound 1 1h and 13c-NMR spectrum.
Figure 10 shows the HPLC data for compound 2 quantitative analyses.
Figure 11 shows compound 2 1h and 13c-NMR spectrum.
Figure 12 shows the HPLC data for compound 3 quantitative analyses.
Figure 13 shows compound 3 1h and 13c-NMR spectrum.
Figure 14 shows the HPLC data for compound 4 quantitative analyses.
Figure 15 shows compound 4 1h and 13c-NMR spectrum.
Figure 16 shows the HPLC data for compound 5 quantitative analyses.
Figure 17 shows compound 5 1h and 13c-NMR spectrum.
Figure 18 shows the HPLC data for compound 6 quantitative analyses.
Figure 19 shows compound 6 1h and 13c-NMR spectrum.
Figure 20 shows the HPLC data for compound 7 quantitative analyses.
Figure 21 shows compound 7 1h and 13c-NMR spectrum.
Figure 22 shows the HPLC data for compound 8 quantitative analyses.
Figure 23 shows compound 8 1h and 13c-NMR spectrum.
Figure 24 shows the HPLC data for compound 9 quantitative analyses.
Figure 25 shows compound 9 1h and 13c-NMR spectrum.
Figure 26 is for showing the figure of the vigor of the B16F10 cell of using Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and solvent section processes thereof.Data are three mean+SD of measuring.
Figure 27 is for showing the figure of the inhibition activity that Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and solvent part thereof generate melanin in B16F10 cell.Data are three mean+SD of measuring.
Figure 28 is for showing Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and the figure of solvent part to the inhibition activity of tryrosinase in B16F10 cell thereof.Data are three mean+SD of measuring.
Figure 29 is for showing the figure of the vigor of the B16F10 cell of processing with lucidone.Data are three mean+SD of measuring.
Figure 30 is for showing the figure of the vigor of the B16F10 cell of processing with linderone methyl ether (methyllinderone).Data are three mean+SD of measuring.
Figure 31 is for showing the figure of the vigor of the B16F10 cell of processing with Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative (kanakugiol).Data are three mean+SD of measuring.
Figure 32 is for showing the figure of the inhibition activity that lucidone generates melanin in B16F10 cell.Data are three mean+SD of measuring.
Figure 33 is for showing the figure of the inhibition activity that linderone methyl ether generates melanin in B16F10 cell.Data are three mean+SD of measuring.
Figure 34 is for showing the figure of the inhibition activity that Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative generates melanin in B16F10 cell.Data are three mean+SD of measuring.
Figure 35 is for showing the figure of lucidone to the inhibition activity of tryrosinase in B16F10 cell.Data are three mean+SD of measuring.
Figure 36 is for showing the figure use from the vigor of the B16F10 cell of the compound treatment of the Chemical formula 10 of Fructus Pyracanthae Fructus Linderae Glaucae separation.
Figure 37 is for showing the figure of inhibition activity melanin B16F10 cell being generated from the compound of the Chemical formula 10 of Fructus Pyracanthae Fructus Linderae Glaucae separation.
Figure 38 shows from the compound of the Chemical formula 10 of Fructus Pyracanthae Fructus Linderae Glaucae separation active to the inhibition of tryrosinase, TRP-1 and TRP-2 gene expression B16F10 cell.C and Arb represent contrast and arbutin.
The mode of invention
Can understand better the present invention by following examples, following examples are in order to illustrate, and should not be construed as restriction the present invention.
Embodiment 1: the preparation of Fructus Pyracanthae Fructus Linderae Glaucae extract
For the vegetable material using, by living resources extract storehouse, Jizhou (JejuBio-Resource Extract Bank), obtain Fructus Pyracanthae Fructus Linderae Glaucae.
First, with flowing water washing tree material (Fructus Pyracanthae Fructus Linderae Glaucae), with hot blast, at 40 ℃, be dried three days and use pulverizer to pulverize.Under room temperature, the dry material of 200g is immersed in 70% ethanol three days and follow and stir so that exudate to be provided.This exudate is filtered.To ooze out with filter process in triplicate.Gained filter liquor is carried out to concentrating under reduced pressure, thereby obtain the ethanol extraction of 60g.
Embodiment 2: from the separated solvent part of Fructus Pyracanthae Fructus Linderae Glaucae extract
In this embodiment for the separated solvent of part purchased from Merk Co., Junsei Co. and Hyman Co..
The ethanol extraction of Fructus Pyracanthae Fructus Linderae Glaucae is carried out to the separation of following solvent part:
First, the 42g in the 60g Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction (70% ethanol extraction) of preparation in embodiment 1 is suspended in distilled water, and use separatory funnel, utilize normal hexane (n-Hex), dichloromethane (CH 2cl 2), that ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) carry out part is separated.Filter subsequently and concentrating under reduced pressure, the amount that obtains each solvent part is as follows: with n-Hex, be 8.56g, use CH 2cl 2for 1.47g, be 4.14g, be 10.2g and use H with n-BuOH with EtOAc 2o is 14.58g (referring to Fig. 1).
Each extract and part are carried out to HPLC analysis, and in Fig. 2 to 7, provide result.
In subsequent experimental, the hexane part of powder type, dichloromethane part or ethyl acetate are partly dissolved in 100% ethanol, butanols part or water section are dissolved in to 100% ethanol: 1 * phosphate buffer (PBS simultaneously, pH 7.4) be in the mixture of 1: 1, before using, filter subsequently.
Embodiment 3: separation and authenticating compound from the solvent part of Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction
Use kieselguhr, to utilize dichloromethane, ether, ethyl acetate and acetone that ethyl acetate part layer (4.0g) is carried out to part more separated.Use chloroform and methanol, as developing solvent, the ether part in gained part is divided into subdivision in purification on normal-phase silica gel.Use purification on normal-phase silica gel, utilize chloroform and methanol that part-5 are wherein further purified.Respectively from part-1 and separating compound 1 and 2-3 partly.Use preparation HPLC, utilize 30% methanol solution that part-2 are further purified, with separating compound 3 and 4.
Use purification on normal-phase silica gel, utilize the solvent gradient of hexane and ethyl acetate that dichloromethane part layer (2.53g) is further divided into 10 subdivisions.Use purification on normal-phase silica gel, utilize hexane and ethyl acetate to incite somebody to action part-2 purification with separating compound 5 as developing solvent.Use purification on normal-phase silica gel, utilize chloroform and methanol as developing solvent separating compound 6 from part-5.Respectively by part-6 and part-7 recrystallization so that compound 7 and 8 to be provided.Use chloroform and methanol, as developing solvent, purification on normal-phase silica gel chromatography is carried out to so that two parts to be provided in part-8.By part-8-2 wherein for separating of compound 9.When analyzing by HPLC and NMR, it is pure by nine kinds of compounds of above acquisition, being proved to be.
In brief, the leaf with the Fructus Pyracanthae Fructus Linderae Glaucae of 70% ethanol extraction 200g, has obtained 60g extract, from 60g extract separated coldest days of the year end kind compound.
Separated part and the yield of compound in following table 1, have been summed up.
The yield of the extract of table 1. Fructus Pyracanthae Fructus Linderae Glaucae and part thereof
Material Yield (%)
Normal hexane part 20.400
Dichloromethane part 6.000
Ethyl acetate part 9.800
N-butyl alcohol part 24.200
Water section 34.700
Compound 1 0.600
Compound 2 0.060
Compound 3 0.002
Compound 4 0.720
Compound 5 0.020
Compound 6 0.030
Compound 7 0.240
Compound 8 0.090
Compound 9 0.110
Use NMR instrument (Bruker Co.500MHz, NMR) obtains compound 1h, 13c, COSY, HMQC, HMBC spectrum, and analyze the structure with deterministic compound.Carry out high performance liquid chromatography (HPLC) for quantitative analysis.
In Fig. 8 to 25, provided compound HPLC result and 1h, 13c-NMR spectrum.
Therefore, compound 1 is accredited as the Quercetin with following Chemical formula 1:
[Chemical formula 1]
Compound 2 is accredited as the Quercitroside with following Chemical formula 2:
[Chemical formula 2]
Compound 3 is accredited as kaempferol-3-O-Fructus rhamni (Rhamnus davurica Pall.) pyranoside with following chemical formula 3:
[chemical formula 3]
Compound 4 is accredited as Quercetin-3-O-arabinofuranosyl glycosides with following chemical formula 4:
[chemical formula 4]
Compound 5 is accredited as the caffeic acetate with following chemical formula 5:
[chemical formula 5]
Compound 6 is accredited as the methyl cinnamate with following chemical formula 6:
[chemical formula 6]
Compound 7 is accredited as the lucidone with following chemical formula 7:
[chemical formula 7]
Compound 8 is accredited as the linderone methyl ether with following chemical formula 8:
[chemical formula 8]
Compound 9 is accredited as the Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative with following chemical formula 9:
[chemical formula 9]
EXPERIMENTAL EXAMPLE 1: the Antioxidative Activity Determination of Fructus Pyracanthae Fructus Linderae Glaucae extract and part
1) 1,1-diphenyl-2-picryl hydrazine (DPPH) free radical scavenging is measured
According to Blosis method, by DPPH free radical scavenging, measure electron donation.By the sample aliquot of variable concentrations in ethanol, be every hole 100 μ L, and the 0.4mMDPPH solution of same volume is also added in 96 orifice plates, under room temperature standing 10 minutes, then under 517nm, measure absorbance.Butylatedhydroxyanisole (BHA) is used as to positive control.Concentration (the IC of sample when calculating DPPH free radical scavenging activity and the absorbance that activity is expressed as DPPH is reduced to 50% according to following formula 1 50).
Formula 1
DPPH free radical scavenging activity (%)=(A contrast-A sample)/A contrast* 100
Wherein
A sample=the absorbance of solution that comprises sample,
A contrast=comprise ethanol and do not comprise the absorbance of the solution of sample.
The measurement result that has shown the antioxidant activity of Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and further part thereof in following table 2.For ethanol extraction and further part thereof, the free radical scavenging activity of DPPH increases in dose-dependent mode.In further part, ethyl acetate partly makes DPPH show the free radical scavenging activity higher than contrast BHA.Except dichloromethane part and hexane part, observe other parts and show remarkable high activity, IC 50value is 7.43 (ethyl acetate), 16.88 (ethanol), 18.54 (butanols) and 66.77 μ g/mL (water) (table 2).
2) xanthine oxidase inhibitory activity and superoxides are removed active mensuration
With allopurinol (Sigma) in contrast, the uric acid of measuring by xanthine oxidase by the absorbance increasing under 290nm produces.Use blue (NBT) reducing process of tetrazole under 560nm, to measure the amount of superoxides.By dissolve sample, 0.5mM xanthine and the 1mM EDTA of each concentration in 100 μ L 200mM phosphate buffers (pH 7.5), carry out preparation feedback solution.In order to induce the generation of uric acid, 50mU/ml xanthine oxidase is added in reaction solution.In order to measure superoxides, remove activity, 0.5mM NBT is added in reaction solution.Concentration (the IC of sample when xanthine oxidase inhibitory activity and superoxides are removed to the active uric acid that is expressed as formation and superoxides and reduced by 50% 50).Each sample is measured in triplicate and is calculated the meansigma methods of measuring for three times.
In table 2, shown that the ethanol extraction of Fructus Pyracanthae Fructus Linderae Glaucae and the xanthine oxidase inhibitory activity of further part thereof and superoxide radical remove active measurement result.All Fructus Pyracanthae Fructus Linderae Glaucae ethanol extractions and further part thereof show the xanthine oxidase inhibitory activity that concentration relies on mode.Although at hexane part (> 1000 μ g/mL) and dichloromethane part (IC 5086.21 μ g/mL) in, low activity detected, but other parts show high activity, the IC of suppressing 50value is 5.55 μ g/mL to 9.79 μ g/mL (table 2).For superoxides, remove activity, IC detected 50value is 16.86 μ g/mL (ethyl acetate parts), and this is than contrast allopurinol (IC 50=3.82 μ g/mL) slightly low (table 2).
As previous, report, lipid oxidation and oxidative stress and Scavenger of ROS are relevant to stoping, antioxidant activity causes prevention or slow down aging or blocking-up cancer and cardiopathic generation or progress, and has application prospect in a plurality of fields that comprise food industry, pharmaceutical industries and cosmetics industry.Therefore, consider the antioxidant activity of Fructus Pyracanthae Fructus Linderae Glaucae, it can be used in and prevents esoteric various oxidative stress and disease.
Table 2
EXPERIMENTAL EXAMPLE 2: the cytotoxicity MTT of Fructus Pyracanthae Fructus Linderae Glaucae extract and part measures
Use in the present embodiment the B16F10 mouse melanin tumor cell of buying from American type culture collection (ATCC).At 5%CO 2in incubator, at 37 ℃, in the DMEM (Gibco) of hyclone (FBS, Gibco), 1% antibiotic-antifongin (Gibco), L-glutaminate and sodium bicarbonate containing 10%, cultivate B16F10 cell.In addition, from KCLB (Korea S's cell line storehouse), buy RAW 264.7 cells of mouse macrophage, and at 5%CO 2in incubator, at 37 ℃, containing in the DMEM (Gibco) of 10%FBS, 100 units/mL penicillin and 100 μ g/mL streptomycins, cultivating.
In 24 orifice plates with 2 * 10 4the density inoculation B16F10 cell of cells/well at 5%CO 2in incubator, at 37 ℃, cultivate 24 hours, then hatch 72 hours with the sample of every hole 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL concentration.The 2mg/mLMTT of 200 μ L is added into cell.Hatch under the same conditions after 4 hours, culture medium is removed and with PBS by twice of cell washing.To the DMSO that adds 200 μ L in every hole, then by ELISA analyzer (μ Quant, USA), measure the absorbance under 570nm.
In order to detect the impact on B16F10 cell line vigor of Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and further part thereof, the extract of cell and 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL variable concentrations and part are hatched three days.Then, by MTT, measure cell viability.As shown in Figure 2, under the concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL of extract and part, observe the 81%-103% that cell viability is control cells vigor, and therefore slightly high or slightly lower than contrast.Because when using the concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL, they do not cause the remarkable change of cell viability, Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction of the present invention and further part thereof are low cytotoxicity and the composition that therefore can be used as skin whitener.
EXPERIMENTAL EXAMPLE 3: Fructus Pyracanthae Fructus Linderae Glaucae extract and part are for the mensuration of the inhibition activity of melanin biosynthesis
In 24 orifice plates with 2 * 10 4the density inoculation B16F10 cell of cells/well at 5%CO 2in incubator, at 37 ℃, cultivate 24 hours, hatch three days with the sample of every hole 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL concentration subsequently.Culture medium is removed and with PBS by twice of cell washing.To the 1N NaOH that adds 500 μ L in every hole, at 56 ℃, hatch subsequently 30 minutes with dissolved cell.Use ELISA analyzer (μ Quant, USA) to read the absorbance under 405nm.
Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and further part thereof are applied to cell after three days with the variable concentrations of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL, measure its inhibition to melanin biosynthesis active, it is avirulent that the Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction of above-mentioned concentration and further part thereof are all observed cell in EXPERIMENTAL EXAMPLE 2.As shown in Figure 3, when using the concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL, Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction shows respectively 0.6%, 7.9%, 31.6% and 45.8% melanin inhibition, and when using the known contrast arbutin as skin whitener of concentration of 100 μ g/mL, the inhibition that arbutin produces melanin is 31%.Meanwhile, concentration is that the further part of 100 μ g/mL shows respectively 33%, 49%, 24%, 31% and 28% the inhibition to melanin biosynthesis, and this is better than or is active similar in appearance to the inhibition of contrast.Because Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction or its further part have good inhibition activity and the low cytotoxicity to melanin biosynthesis, so they can be used as natural skin brightening agent.
EXPERIMENTAL EXAMPLE 4: Fructus Pyracanthae Fructus Linderae Glaucae extract and the part mensuration to the inhibition activity of tryrosinase
In 24 orifice plates with 2 * 10 4the density inoculation B16F10 cell of cells/well at 5%CO 2in incubator, at 37 ℃, cultivate 24 hours, hatch three days with the sample of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL concentration subsequently.After culture medium is removed, with PBS, by twice of cell washing centrifugal so that cell precipitation group to be provided, then to cell precipitation group, add lysis buffer (0.1M sodium phosphate buffer, 0.2mM PMSF, 1% triton x-100).Cell in lysis buffer is placed in and carries out lysis on ice.After centrifugal, supernatant is used for measuring enzymatic activity.Each sample is added in reaction buffer to (12.5mM L-3,4 dihydroxyphenylalanine, 1.5mM TYR, 67mM sodium phosphate buffer (pH6.8)) and at 37 ℃, hatches 1 hour, then, use ELISA analyzer (μ Quant, USA) to measure absorbance under 405nm.
After processing with Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and further part, melanin terminal level's reduction has reflected the active reduction that participates in the synthetic enzyme of melanin.As shown in Figure 4, when the sample treatment B16F10 cell of the variable concentrations with 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL is in the time of three days, ethanol extraction shows respectively the inhibition to tryrosinase up to 13.9%, 15.9%, 36.8% and 42.5%, and contrast arbutin is 19% to the inhibition of tryrosinase.Under the concentration of 100 μ g/mL, part is respectively 40%, 21%, 19%, 41% and 26% to the inhibition of tryrosinase, and this demonstrates the antityrosinase activity that is better than contrast.The reduction of the melanin biosynthesis that in data acknowledgement B16F10, Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction and further part cause is mainly active to the inhibition of tryrosinase owing to them.
EXPERIMENTAL EXAMPLE 5: use MTT to measure the cytotoxicity of the cyclopentanedione compound in measurement Fructus Pyracanthae Fructus Linderae Glaucae source
1) lucidone (compound 1)
In order to detect the impact of one matter on B16F10 cell line vigor that is separated into lucidone, this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days, by MTT, measure cell viability subsequently.As shown in figure 29, under the caffeic acid concentration of 1.25,2.5,5 and 10 μ g/mL, observe the 96%-106% that cell viability is control cells vigor, and therefore slightly high or slightly low with contrast.While using the one matter that is separated into lucidone of the present invention due to concentration when with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, do not cause the remarkable change of cell viability, therefore the one matter that is separated into lucidone of the present invention is low cytotoxicity therefore can be as the composition of skin whitener.
2) linderone methyl ether (compound 2)
In order to detect the impact of one matter on B16F10 cell line vigor that is separated into linderone methyl ether, this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days, by MTT, measure cell viability subsequently.As shown in figure 30, under the caffeic acid concentration of 1.25,2.5,5 and 10 μ g/mL, observe the 93%-99% that cell viability is control cells vigor, and therefore slightly lower than contrast.While using the one matter that is separated into linderone methyl ether of the present invention due to concentration when with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, do not cause the remarkable change of cell viability, therefore the one matter that is separated into linderone methyl ether of the present invention is low cytotoxicity therefore can be as the composition of skin whitener.
3) Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative (compound 3)
In order to detect the impact of one matter on B16F10 cell line vigor that is separated into Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative, this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days, by MTT, measure cell viability subsequently.As shown in figure 31, under the caffeic acid concentration of 1.25,2.5,5 and 10 μ g/mL, observe the 91%-96% that cell viability is control cells vigor, and therefore slightly lower than contrast.While using the one matter that is separated into Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative of the present invention due to concentration when with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, do not cause the remarkable change of cell viability, therefore the one matter that is separated into Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative of the present invention is low cytotoxicity therefore can be as the composition of skin whitener.
EXPERIMENTAL EXAMPLE 6: the mensuration of the cyclopentanedione compound in Fructus Pyracanthae Fructus Linderae Glaucae source to the inhibition activity of melanin biosynthesis
1) lucidone (compound 1)
In order to detect the inhibitory action of one matter to melanin biosynthesis in B16F10 cell line that is separated into lucidone, this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days and analyzed melanin biosynthesis.
Shown in figure 32, when using lucidone with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, it shows respectively the inhibition up to 3%, 19%, 37% and 53%, and when the concentration use with 50 μ g/mL is known as the contrast arbutin of skin whitener, arbutin is 23.5% to the inhibition of melanin biosynthesis.Compare with contrasting arbutin, this compound has the stronger activity of the inhibition to melanin biosynthesis and can be used as natural skin whitener.
2) linderone methyl ether (compound 2)
In order to detect the inhibitory action of one matter to melanin biosynthesis in B16F10 cell line that is separated into linderone methyl ether, this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days and analyzed melanin biosynthesis.
As shown in figure 33, when using linderone methyl ether with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, it shows respectively the inhibition up to 1%, 14%, 38% and 62%, and when the concentration use with 50 μ g/mL is known as the contrast arbutin of skin whitener, arbutin is 23.5% to the inhibition of melanin biosynthesis.Compare with contrasting arbutin, this compound has the stronger activity of the inhibition to melanin biosynthesis and can be used as natural skin whitener.
3) Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative (compound 3)
In order to detect the inhibitory action of one matter to melanin biosynthesis in B16F10 cell line that is separated into Fructus Pyracanthae Fructus Linderae Glaucae chalcone derivative, respectively this compound of cell and 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL variable concentrations is hatched three days and analyzed melanin biosynthesis.
As shown in figure 34, when using lucidone with 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL, it shows respectively the inhibition up to 2%, 16%, 29% and 61%, and when the concentration use with 50 μ g/mL is known as the contrast arbutin of skin whitener, arbutin is 23.5% to the inhibition of melanin biosynthesis.Compare with contrasting arbutin, this compound has the stronger activity of the inhibition to melanin biosynthesis and can be used as natural skin whitener.
EXPERIMENTAL EXAMPLE 7: the mensuration of the cyclopentanedione compound in Fructus Pyracanthae Fructus Linderae Glaucae source to the inhibition activity of tryrosinase
As shown in figure 35, when with the sample treatment B16F10 cell of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL variable concentrations in the time of three days, lucidone shows respectively 10%, 17%, 26.4% and 48.9% the inhibition to tryrosinase, and contrast arbutin is 19% to the inhibition of tryrosinase.Based on these data, think that the reduction of melanin biosynthesis is mainly active owing to its inhibition to tryrosinase the B16F10 causing from the lucidone of Fructus Pyracanthae Fructus Linderae Glaucae separation.
Embodiment 4: from the preparation of the leaf of Fructus Pyracanthae Fructus Linderae Glaucae and the extract of bark and solvent part thereof
The Fructus Pyracanthae Fructus Linderae Glaucae being obtained by living resources extract storehouse, Jizhou is used as to vegetable material.Extracting solvent is the product of Merk Co..
Fresh leaf with flowing water washing Fructus Pyracanthae Fructus Linderae Glaucae is dried three days with hot blast at 40 ℃, and uses pulverizer to pulverize.Under room temperature, the drying material of 200g immersed in 70% ethanol three days and follow stirring, so that exudate to be provided.This exudate is filtered, and will ooze out with filter process in triplicate.By gained filter liquor concentrating under reduced pressure, to obtain 60g ethanol extraction.42g in 60g Fructus Pyracanthae Fructus Linderae Glaucae ethanol extraction (70% ethanol extraction) is suspended in distilled water, and with normal hexane (n-Hex), dichloromethane (CH 2cl 2), that ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) carry out part is separated.Filter and vacuum concentration, the amount of the extract of acquisition is as follows: use n-Hex is 8.56g, use CH 2cl 2for 1.47g, use EtOAc for 4.14g, to use n-BuOH be 10.2g and use H 2o is 14.58g.
In the mode identical with leaf, the bark of Fructus Pyracanthae Fructus Linderae Glaucae is dried and is pulverized.Under the same conditions, use the sample that 560g pulverizes obtain 70% ethanol extraction of 68.2g and carry out part separation with solvent.
Embodiment 5: separation and authenticating compound from the solvent part of Fructus Pyracanthae Fructus Linderae Glaucae
The filler using in the separation of this embodiment is purification on normal-phase silica gel 60 (0.063-0.200mm, Merck Co.).Analytical tool is for being equipped with XTerra the HPLC of C18 3.5 μ m 4.6 * 100mm (Alliance2695, Waters Co.).The detector that PDA Model 2998 use of Waters are performed an analysis.Use solvent and the reagent of HPLC level.NMR (nuclear magnetic resonance, NMR) device for structural analysis is the JNM-LA500 of Burker (German Co.).By methanol-d 4and chloroform-d 1the solvent of analyzing as NMR.
The solvent gradient of use purification on normal-phase silica gel, utilizing hexane/ethyl acetate (v/v) is further divided into 10 subdivisions by the dichloromethane part layer (2.5g) obtaining from the leaf of Fructus Pyracanthae Fructus Linderae Glaucae in embodiment 4.By part-6 wherein, by recrystallization, be further purified to provide compound 10 (21.8mg).
In addition, the solvent gradient of use purification on normal-phase silica gel, utilizing hexane/ethyl acetate (v/v) is further divided into 8 subdivisions by the dichloromethane part layer (7g) obtaining from the bark of Fructus Pyracanthae Fructus Linderae Glaucae in embodiment 4.Part-2 wherein are further placed in to purification on normal-phase silica gel (hexane/ethyl acetate is 3/2) so that part to be provided.By part-2-2 wherein for separating of compound 12 (59.6mg), separating compound 11 from part-2-3 simultaneously.
By the structure of HPLC, NMR (1D, 2D NMR) and LR/HR FAB-MS analysis of compounds 10 to 12, and by HPLC, analyze purity and the content of compound 10 to 12 in ethanol extraction.
As a result, compound 10 is accredited as lucidone, is cyclopentanedione; Compound 11 is accredited as linderone methyl ether, is also cyclopentanedione; Compound 12 is accredited as the cyclopentanedione derivant of the modification being represented by following Chemical formula 10, the noval chemical compound (N2) that it is not reported before being.The IUPAC name of this noval chemical compound is called 2,3,4,5-tetramethoxy-6-(1-methoxyl group-3-phenylpropyl) phenol], and be called as Jizhou-Polymethoxylated-phenol.1D and the 2D NMR data of compound 12 are provided in following table 3.
[Chemical formula 1 0]
Character:
1) IUPAC name: 2,3,4,5-tetramethoxy-6-(1-methoxyl group-3-phenylpropyl) phenol;
2) color: bottle green;
3) abnormal smells from the patient: slight corrupt abnormal smells from the patient;
4) dissolubility: be dissolved in the organic solvent such as methanol, acetone, chloroform etc.;
5) be only distributed in the bark of Fructus Pyracanthae Fructus Linderae Glaucae;
6) common name: Jizhou-Polymethoxylated-phenol.
The 1D of table 3. compound 12 and 2D NMR data
In following table 4, summed up the yield from the compound of the leaf of Fructus Pyracanthae Fructus Linderae Glaucae and the ethanol extraction of bark, solvent part and Chemical formula 10.
Table 4
As shown in table 4, the yield of the compound of finding Chemical formula 10 in bark be up to 0.71%, and in leaf, do not find this compound.
To be dissolved in DMSO completely from the single compound of Fructus Pyracanthae Fructus Linderae Glaucae separation, and filter for subsequent experimental.
EXPERIMENTAL EXAMPLE 8: the Cytotoxic MTT of the compound of Chemical formula 10 measures
According to below, to so far not report noval chemical compound in B16F10 cell line, carry out toxicity detection.
Use in the present embodiment the B16F10 mouse melanin tumor cell of buying from American type culture collection (ATCC).At 5%CO 2in incubator, at 37 ℃, in the DMEM (Gibco) containing 10% hyclone (FBS, Gibco), 1% antibiotic-antifongin (Gibco), L-glutaminate and sodium bicarbonate, cultivate B16F10 cell.
In 24 orifice plates with 2 * 10 4the density inoculation B16F10 cell of cells/well, and at 5%CO 2in incubator, at 37 ℃, cultivate 24 hours, then hatch 72 hours with the sample of 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 30 μ g/mL concentration.The 2mg/mLMTT of 200 μ L is added into cell.Hatch under the same conditions after 4 hours, culture medium is removed and with PBS by twice of cell washing.To the DMSO that adds 200 μ L in every hole, then utilize ELISA analyzer (μ Quant, USA) to measure the absorbance under 570nm.
As shown in figure 36, under the concentration of 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 30 μ g/mL of extract and part, observe the 97%-103% that cell viability is control cells vigor, and therefore slightly high or slightly lower than contrast.Owing to not causing the remarkable change of cell viability when using the compound of Chemical formula 10 of the present invention with the concentration of 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 30 μ g/mL, therefore the compound of Chemical formula 10 of the present invention is low cytotoxicity and the composition that therefore can be used as skin whitener.
EXPERIMENTAL EXAMPLE 9: the mensuration of the compound of Chemical formula 10 to the inhibition activity of melanin biosynthesis
In 24 orifice plates with 2 * 10 4the density inoculation B16F10 cell of cells/well, and at 5%CO 2in incubator, at 37 ℃, cultivate 24 hours, hatch three days with sample subsequently.Culture medium is removed and with PBS by twice of cell washing.To the 1N NaOH that adds 500 μ L in every hole,, at 56 ℃, hatch 30 minutes with dissolved cell, then with ELISA analyzer (μ Quant, USA), measuring the absorbance under 405nm.
Therefore, as shown in figure 37, when using noval chemical compound of the present invention with the concentration of 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 30 μ g/mL, it shows respectively the inhibition of 19%, 32%, 51% and 59% melanin biosynthesis, and when usining the concentration of 50 μ g/mL while using the known contrast arbutin as skin whitener, arbutin is 33.6% to the inhibition of melanin biosynthesis.Because noval chemical compound of the present invention has the good inhibition activity to melanin biosynthesis for 2 times of contrast arbutin, therefore can be used as natural skin brightening agent.
EXPERIMENTAL EXAMPLE 10: the mensuration of the compound of Chemical formula 10 to the inhibition activity of the mrna expression of the gene of responsible melanin biosynthesis
Use in the present embodiment the B16F10 mouse melanin tumor cell of buying from American type culture collection (ATCC).At 5%CO 2in incubator, at 37 ℃, in the DMEM (Gibco) containing 10% hyclone (FBS, Gibco), 1% antibiotic-antifongin (Gibco), L-glutaminate and sodium bicarbonate, cultivate B16F10 cell.
After cultivation, by PBS washed twice or three times for cell, and it is separated to use TRIzol reagent (Invitrogen, USA) to carry out total RNA to cell.With TRIzol-reagent, by cell homogenization and with chloroform, mix, then centrifugal (12,000rpm, 15 minutes).To the isopropyl alcohol that adds same volume in supernatant, then centrifugal (12,000rpm, 10 minutes) are with precipitated rna.The washing with alcohol RNA precipitate of processing with 75% pyrocarbonic acid diethyl ester (DEPC), is dried and is dissolved in the water of DEPC processing.By the absorbance under measurement 260nm, determine the amount of RNA.By A260/A280nm ratio, be that unique RNA component of 1.6 to 1.9 is for reverse transcription.By means of Improm-II tMit is synthetic that cDNA test kit (kit) (Promega, USA) carries out cDNA.In this process, at 1 RNA of unit enzyme inhibitor and Improm-II tMunder the existence of reverse transcription (2U), total RNA of 1 μ g separation, widow's (dT) primer and dNTP (0.5 μ M) are heated 5 minutes in 25 ℃, then at 37 ℃, heat 60 minutes, then by heating, within 10 minutes, stop reverse transcription at 70 ℃, thus synthetic cDNA.By polymerase chain reaction (PCR) from synthetic cDNA amplification tryrosinase, TRP-1, TRP-2 and beta-actin.For this process, by 1 μ L synthetic cDNA, 4 μ M 5 '-and 3 '-primer, 10 * buffer (10mMTris-HCl, pH 8.3,50mM KCl, 0.1% triton x-100), 250 μ M dNTP, 25mM MgCl 2mix to form the cumulative volume of 25 μ L with deionized water with 1 Taq of unit polymerase (Promega, USA), and by it at the enterprising performing PCR of Perkin-Elmer thermal cycler.PCR carries out 20 to 25 circulations, and each circulation is: 94 ℃/30 seconds, 50 ℃-55 ℃/45 seconds and 72 ℃/45 seconds.Thus obtained PCR product is carried out to electrophoresis on 1.2% agarose gel and use ethidium bromide staining to show particular bands.
In melanocyte, the NO that keratinocyte produces in response to UV irradiates increases melanin by cGMP approach and generates.In addition, known melanocyte can increase the expression of tryrosinase and TRP-1, and induces the expression of tryrosinase mRNA.In order to detect inhibitory action melanin B16F10 mouse melanin tumor cell being generated from the single compound of Fructus Pyracanthae Fructus Linderae Glaucae separation, whether come from the inhibition to mrna expression, thereby carry out RT-PCR.The expression of the known TRP-1 playing an important role in melanin generates and TRP-2 and tyrosinase cdna is suppressed.
In this context, as shown in figure 38, the mode that tryrosinase and TRP-1 mRNA level rely on concentration reduces.In the synthetic process of melanin, it is reported, the function of TRP-2 is that dopachrome is converted into carboxylic acid derivant 5,6-dihydroxy indole-2-carboxylic acid (DHICA).Yet the expression of observing TRP-2 mRNA remains on constant level, irrelevant with sample concentration.
Therefore,, find by reducing the mrna expression of tryrosinase and TRP-1, to come check melanin to generate from the new compound of Fructus Pyracanthae Fructus Linderae Glaucae separation this mechanism and TRP-2 have nothing to do (Figure 38).In a word, the data acknowledgement of above acquisition has the strong inhibitory activity to melanin generation and tryrosinase B16F10 melanoma cell from the new compound of Fructus Pyracanthae Fructus Linderae Glaucae separation, and therefore can be as the functional components of skin whitening.
Industrial applicibility
As shown in embodiment and EXPERIMENTAL EXAMPLE, discovery comprises from the skin whitening composition of the present invention of the extract of Fructus Pyracanthae Fructus Linderae Glaucae separation, part or compound and has the cyclopentanedione compound or derivatives thereof as active component, and height check melanin generates and tyrosinase activity thus.Therefore, the present invention can be applicable to comprise a plurality of fields of cosmetics industry, pharmaceutical industries and food industry.

Claims (7)

1. following chemical formula 9 and the purposes of 10 represented compounds in preparing skin whitening composition:
[chemical formula 9]
[Chemical formula 1 0]
2. purposes as claimed in claim 1, wherein said compound is separated and purification from Fructus Pyracanthae Fructus Linderae Glaucae (Lindera erythrocarpa).
3. by the represented compound of following Chemical formula 10:
[Chemical formula 1 0]
4. compound as claimed in claim 3, the compound of wherein said Chemical formula 10 is from the separation of Fructus Pyracanthae Fructus Linderae Glaucae.
5. compound as claimed in claim 3, the compound of wherein said Chemical formula 10 is from the bark separation of Fructus Pyracanthae Fructus Linderae Glaucae.
6. from the method for the compound of the separated claim 3 of Fructus Pyracanthae Fructus Linderae Glaucae (Lindera erythrocarpa):
Fructus Pyracanthae Fructus Linderae Glaucae is immersed in solvent so that the extract from Fructus Pyracanthae Fructus Linderae Glaucae to be provided, and described solvent is selected from water, C1 to C4 lower alcohol and combination thereof;
With hexane, dichloromethane, ethyl acetate and butanols by described Fructus Pyracanthae Fructus Linderae Glaucae extract part, so that minute other solvent part to be provided;
Use hexane and ethyl acetate gradient as eluent, described Fructus Pyracanthae Fructus Linderae Glaucae solvent is partly carried out to elementary silica gel column chromatography so that chromatography part to be provided; And
Use the mixture of hexane and ethyl acetate as eluent, make described elementary silica gel column chromatography part carry out purification by secondary silica gel column chromatography, so that new polymethoxy phenolic compounds to be provided.
7. method as claimed in claim 6, wherein said Fructus Pyracanthae Fructus Linderae Glaucae is the bark of Fructus Pyracanthae Fructus Linderae Glaucae.
CN200980141207.8A 2008-09-04 2009-09-04 Skin-whitening composition comprising an extract, fraction or compound derived from lindera erythrocarpa Expired - Fee Related CN102186457B (en)

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